Highly sensitive HIV RNA assays have demonstrated that viral replication persists in patients with clinically suppressed viral replication in their plasma. A number of anatomic sites have been proposed as the source of persistent viral production including the gastrointestinal tract, the central nervous system, lymphoid tissue, and the genitourinary system. Humanized mice and nonhuman primates are models for HIV infection, persistence, and eradication, yet no data currently exist to evaluate inter-species similarities or differences in drug distribution to these sites. This causes difficulty in applying research findings from one species to another, including extrapolating to humans. In addition, no information on intracellular drug distribution within the aforementioned anatomic tissues has been generated. Finally, there is limited information on factors responsible for drug distribution and activity at these sites, including protein binding, drug transporter activity, and (in the caseof nucleoside analogue reverse transcriptase inhibitors) intracellular endogenous nucleotide concentrations. The hypothesis for this project is: residual active viral reservoirs are a consequence of reduced antiretroviral penetration into target tissue cell subtypes, and a thorough understanding of tissue drug distribution between species must be conducted before rational intensification regimens can be designed. Three specific aims are proposed: 1) In tissues implicated as active reservoirs, characterize distribution of 6 commonly used antiretrovirals from 5 drug classes in models of infected and uninfected humanized mice, nonhuman primates, and HIV+ subjects 2) Investigate important physiologic factors that may be responsible for antiretroviral activity and distribution, including protein binding, intracellular endogenous nucleotide concentrations, and drug transporter expression. 3) Develop a novel IR-MALDESI-MSI approach to visualize the dispersion of drugs across tissue reservoirs for all three species, and assess the role of this technology for future tissue studies in HIV infection and therapy. The goal of this work is to identify what species differences and pharmacologic barriers exist in extracellular and intracellular antiretroviral biodistribution and efficacy in eliminating active HIV reservoirs. Data generated from this proposal will enable the rational selection of antiretrovirals well suited to target active reservoirs, determine the pharmacologic advantages and limitations of animal models of HIV infection, and develop a novel strategy to measure cross-sectional drug dispersion in tissues.